Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells.

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.

Resolvin D2 attenuates LPS-induced macrophage exhaustion.

Early in sepsis, a hyperinflammatory response is dominant, but later, an immunosuppressive phase dominates, and the host is susceptible to opportunistic infections. Anti-inflammatory agents may accelerate the host into immunosuppression, and few agents can reverse immunosuppression without causing inflammation. Specialized pro-resolving mediators (SPMs) such as resolvin D2 (RvD2) have been reported to resolve inflammation without being immunosuppressive, but little work has been conducted to examine their effects on immunosuppression. To assess the effects of RvD2 on immunosuppression, we established a model of macrophage exhaustion using two lipopolysaccharide (LPS) treatments or hits. THP-1 monocyte-derived macrophages were first treated with RvD2 or vehicle for 1 h. One LPS hit increased NF-κB activity 11-fold and TNF-α release 60-fold compared to unstimulated macrophages. RvD2 decreased LPS-induced NF-κB activity and TNF-α production but increased bacterial clearance. Two LPS hits reduced macrophage bacterial clearance and decreased macrophage NF-κB activity (45%) and TNF-α release (75%) compared to one LPS hit, demonstrating exhaustion. RvD2 increased NF-κB activity, TNF-α release, and bacterial clearance following two LPS hits compared to controls. TLR2 inhibition abolished RvD2-mediated changes. In a mouse sepsis model, splenic macrophage response to exogenous LPS was reduced compared to controls and was restored by in vivo administration of RvD2, supporting the in vitro results. If RvD2 was added to monocytes before differentiation into macrophages, however, RvD2 reduced LPS responses and increased bacterial clearance following both one and two LPS hits. The results show that RvD2 attenuated macrophage suppression in vitro and in vivo and that this effect was macrophage-specific.

Protocols for Co-Culture Phenotypic Assays with Breast Cancer Cells and THP-1-Derived Macrophages.

Tumor mass comprises not only cancer cells but also heterogeneous populations of immune and stromal cells, along with the components of the extracellular matrix, collectively called the tumor microenvironment (TME). This diverse population of cells can communicate with each other, which can positively or negatively affect tumor growth and progression to malignancy. The most common type of immune cells in the TME are macrophages. Macrophages continuously differentiate into a broad landscape of tumor-associated macrophages (TAMs) in response to numerous signals from the TME, which makes studies on TAMs quite challenging. Therefore, implementing reliable protocols is a milestone for drawing consistent conclusions about the interactions between cancer cells and TAMs. Here, we provide the details for the polarization of a human leukemia monocytic cell line, THP-1, into M0, M1 and M2 macrophages. We also present a step-by-step protocol for a transwell co-culture using a human breast cancer cell line, HCC1806, and THP-1-derived macrophages. Finally, we describe the colony formation and migration assays performed on the breast cancer cells after the co-culture with macrophages to measure the influence of macrophages on the oncogenic features of cancer cells. In summary, our co-culture-based protocols can be a valuable resource for investigating the interactions between macrophages and cancer cells.

The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) inhibits HIV-1 replication in nondividing cells by reducing the intracellular dNTP pool. SAMHD1 also suppresses NF-κB activation induced by inflammatory stimuli and viral infections. Specifically, SAMHD1-mediated reduction of NF-κB inhibitory protein (IκBα) phosphorylation is important for the suppression of NF-κB activation. However, while the inhibitors of NF-κB kinase subunit alpha and beta (IKKα and IKKß) regulate IκBα phosphorylation, the mechanism by which SAMHD1 regulates phosphorylation of IκBα remains unclear. Here, we report that SAMHD1 suppresses phosphorylation of IKKα/ß/γ via interaction with IKKα and IKKß, thus inhibiting subsequent phosphorylation of IκBα in monocytic THP-1 cells and differentiated nondividing THP-1 cells. We show that knockout of SAMHD1 enhanced phosphorylation of IKKα, IKKß, and IKKγ in THP-1 cells treated with the NF-κB activator lipopolysaccharide or infected with Sendai virus and SAMHD1 reconstitution inhibited phosphorylation of IKKα/ß/γ in Sendai virus-infected THP-1 cells. We demonstrate that endogenous SAMHD1 interacted with IKKα and IKKß in THP-1 cells and recombinant SAMHD1 bound to purified IKKα or IKKß directly in vitro. Mapping of these protein interactions showed that the HD domain of SAMHD1 interacts with both IKKα and IKKß and that the kinase domain of IKKα and the ubiquitin-like domain of IKKß are required for their interactions with SAMHD1, respectively. Moreover, we found that SAMHD1 disrupts the interaction between upstream kinase TAK1 and IKKα or IKKß. Our findings identify a new regulatory mechanism by which SAMHD1 inhibits phosphorylation of IκBα and NF-κB activation.

Antibodies from chlamydia-infected individuals facilitate phagocytosis via Fc receptors.

Non-neutralizing functions of antibodies, including phagocytosis, may play a role in Chlamydia trachomatis (CT) infection, but these functions have not been studied and assays are lacking. We utilized a flow-cytometry-based assay to determine whether serum samples from a well-characterized cohort of CT-infected and naïve control individuals enhanced phagocytosis via Fc-receptor-expressing THP-1 cells, and whether this activity correlated with antibody titers. Fc-receptor-mediated phagocytosis was detected only in CT+ donors. Phagocytosis generally did not correlate well with antibody titer. In addition, we found that complement from both CT+ and negative individuals enhanced phagocytosis of CT into primary neutrophils. These results suggest that anti-CT antibodies can have functions that are not reflected by titer. This method could be used to quantitively measure Fc-receptor-mediated function of anti-CT antibodies or complement activity and could reveal new immune correlates of protection.

Specific interaction between Group B Streptococcus CC17 hypervirulent clone and phagocytes.

Streptococcus agalactiae also named Group B Streptococcus (GBS) is the most significant pathogen causing invasive infections, such as bacteremia and meningitis, in neonates. Worldwide epidemiological studies have shown that a particular clonal complex (CC) of capsular serotype III, the CC17, is strongly associated with meningitis in neonates and is therefore, designated as the hypervirulent clone. Macrophages are a permissive niche for intracellular bacteria of all GBS clones. In this study, we deciphered the specific interaction of GBS CC17 strains with macrophages. Our study revealed that CC17 strains are phagocytosed at a higher rate than GBS non-CC17 strains by human monocytes and macrophages both in cellular models and in primary cells. CC17-enhanced phagocytosis is due to an initial enhanced-attachment step to macrophages mediated by the CC17-specific surface protein HvgA and the PI-2b pilus (Spb1). We showed that two different inhibitors of scavenger receptors (fucoidan and poly(I)) specifically inhibited CC17 adhesion and phagocytosis while not affecting those of non-CC17 strains. Once phagocytosed, both CC17 and non-CC17 strains remained in a LAMP-1 positive vacuole that ultimately fuses with lysosomes where they can survive at similar rates. Finally, both strains displayed a basal egress which occurs independently from actin and microtubule networks. Our findings provide new insights into the interplay between the hypervirulent GBS CC17 and major players of the host's innate immune response. This enhanced adhesion, leading to increased phagocytosis, could reflect a peculiar capacity of the CC17 lineage to subvert the host immune defenses, establish a niche for persistence or disseminate.

The dual role of FSP1 in programmed cell death: resisting ferroptosis in the cell membrane and promoting necroptosis in the nucleus of THP-1 cells.

BACKGROUND: Acute monocytic leukemia-M5 (AML-M5) remains a challenging disease due to its high morbidity and poor prognosis. In addition to the evidence mentioned earlier, several studies have shown that programmed cell death (PCD) serves a critical function in treatment of AML-M5. However, the role and relationship between ferroptosis and necroptosis in AML-M5 remains unclear. METHODS: THP-1 cells were mainly treated with Erastin and IMP-366. The changes of ferroptosis and necroptosis levels were detected by CCK-8, western blot, quantitative real-time PCR, and electron microscopy. Flow cytometry was applied to detect the ROS and lipid ROS levels. MDA, 4-HNE, GSH and GSSG were assessed by ELISA kits. Intracellular distribution of FSP1 was studied by immunofluorescent staining and western blot. RESULTS: The addition of the myristoylation inhibitor IMP-366 to erastin-treated acute monocytic leukemia cell line THP-1 cell not only resulted in greater susceptibility to ferroptosis characterized by lipid peroxidation, glutathione (GSH) depletion and mitochondrial shrinkage, as the FSP1 position on membrane was inhibited, but also increased p-RIPK1 and p-MLKL protein expression, as well as a decrease in caspase-8 expression, and triggered the characteristic necroptosis phenomena, including cytoplasmic translucency, mitochondrial swelling, membranous fractures by FSP1 migration into the nucleus via binding importin α2. It is interesting to note that ferroptosis inhibitor fer-1 reversed necroptosis. CONCLUSION: We demonstrated that inhibition of myristoylation by IMP-366 is capable of switching ferroptosis and ferroptosis-dependent necroptosis in THP-1 cells. In these findings, FSP1-mediated ferroptosis and necroptosis are described as alternative mechanisms of PCD of THP-1 cells, providing potential therapeutic strategies and targets for AML-M5.

CTRP4 ameliorates inflammation, thereby attenuating the interaction between HUVECs and THP-1 monocytes through SIRT6/Nrf2 signaling.

CTRP4, identified as an adipokine, has demonstrated notable anti-inflammatory and anti-obesity effects in various disease models. Consequently, our research sought to explore the impact of CTRP4 on inflammation and the interaction between endothelial cells and monocytes in hyperlipidemic conditions. Using Western blotting, we assessed the expression levels of various proteins in HUVECs and THP-1 monocytes. Our study findings indicate that treatment with CTRP4 effectively mitigated the attachment of THP-1 monocytes to HUVECs. Furthermore, it reduced the expression of adhesion molecules and inflammation indicators in experimental cells exposed to hyperlipidemic conditions. Notably, CTRP4 treatment led to an increase in SIRT6 expression and the nuclear translocation of Nrf2. Interestingly, when SIRT6 or Nrf2 was silenced using siRNA, the positive effects of CTRP4 in HUVECs and THP-1 cells were nullified. Our results suggest that CTRP4 exhibits anti-inflammatory properties, thereby improving the interaction between endothelial cells and monocytes through the SIRT6/Nrf2-dependent pathway. This study provides insights into CTRP4 as a potential therapeutic target for mitigating obesity-related atherosclerosis.

IL-38 alleviates atherogenic responses via SIRT6/HO-1 signaling: A promising strategy against obesity-related atherosclerosis.

Interleukin-38 (IL-38), a member of the IL-1 family, is known for its anti-inflammatory properties mediated through ligand signaling in various disease models. It plays a significant role in atherosclerosis development, forming a theoretical basis for therapeutic strategies. However, the direct effects of IL-38 on atherogenic responses in the vascular endothelium and monocytes remain unclear. In this investigation, IL-38 treatment reduced THP-1 monocyte adhesion to HUVECs, decreased the expression of vascular adhesion molecules, and mitigated inflammation in the presence of palmitate. IL-38 treatment upregulated SIRT6 expression and enhanced autophagy markers such as LC3 conversion and p62 degradation. The effects of IL-38 were nullified by siRNA-mediated suppression of SIRT6 or heme oxygenase-1 (HO-1) in HUVECs and palmitate-treated THP-1 cells. These findings reveal that IL-38 mitigates inflammation through the SIRT6/HO-1 pathway, offering a potential therapeutic approach for addressing obesity-related atherosclerosis.

Novel mechanism of drug resistance triggered by tumor-associated macrophages through Heat Shock Factor-1 activation.

Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism.

Interleukin-10: a novel metabolic inducer of macrophage differentiation and subsequently contributing to improved pregnancy outcomes of mice by orchestrating oxidative phosphorylation metabolism†.

Metabolism regulates the phenotype and function of macrophages. After recruitment to local tissues, monocytes are influenced by the local microenvironment and differentiate into various macrophages depending on different metabolic pathways. However, the metabolic mechanisms underlying decidual macrophage differentiation remain unknown. Interleukin-10 (IL-10) is an important decidual macrophage inducer and promotes oxidative phosphorylation (OXPHOS) of bone marrow-derived macrophages. In this study, we mainly investigate the metabolic changes involved in IL-10-generated macrophages from monocytes using in vitro models. We demonstrate that exposure of monocytes (either peripheral or THP-1) to IL-10 altered the phenotype and function of resultant macrophages that are linked with OXPHOS changes. Interleukin-10 enhanced the mitochondrial complex I and III activity of THP-1 cell-differentiated macrophages and increased the mitochondrial membrane potential, intracellular adenosine triphosphate, and reactive oxygen species levels. Oxidative phosphorylation blockage with oligomycin changed the cell morphology of IL-10-generated macrophages and the expression levels of cytokines, such as transforming growth factor beta, tumor necrosis factor-alpha, interferon gamma, and IL-10, apart from changes in the expression level of the surface markers CD206, CD209, and CD163. Moreover, in vivo IL-10 administration reduced the lipopolysaccharide (LPS)-induced embryo resorption rate, and this effect was diminished when OXPHOS was inhibited, demonstrating that OXPHOS is important for the improved pregnancy outcomes of IL-10 in LPS-induced abortion-prone mice. Our findings provide deep insights into the roles of IL-10 in macrophage biology and pregnancy maintenance. Nevertheless, the direct evidence that OXPHOS is involved in decidual macrophage differentiation needs further investigations.

Establishment of an in vitro model of monocyte-like THP-1 cells for trained immunity induced by bacillus Calmette-Guérin.

BACKGROUND: Mycobacteria bloodstream infections are common in immunocompromised people and usually have disastrous consequences. As the primary phagocytes in the bloodstream, monocytes and neutrophils play critical roles in the fight against bloodstream mycobacteria infections. In contrast to macrophages, the responses of monocytes infected with the mycobacteria have been less investigated. RESULTS: In this study, we first established a protocol for infection of non-adherent monocyte-like THP-1 cells (i.e. without the differentiation induced by phorbol 12-myristate 13-acetate (PMA) by bacillus Calmette-Guérin (BCG). Via the protocol, we were then capable of exploring the global transcriptomic profiles of non-adherent THP-1 cells infected with BCG, and found that NF-κB, MAPK and PI3K-Akt signaling pathways were enhanced, as well as some inflammatory chemokine/cytokine genes (e.g. CCL4, CXCL10, TNF and IL-1ß) were up-regulated. Surprisingly, the Akt-HIF-mTOR signaling pathway was also activated, which induces trained immunity. In this in vitro infection model, increased cytokine responses to lipopolysaccharides (LPS) restimulation, higher cell viability, and decreased Candida albicans loads were observed. CONCLUSIONS: We have first characterized the transcriptomic profiles of BCG-infected non-adherent THP-1 cells, and first developed a trained immunity in vitro model of the cells.

Soluble urate-induced effects on cytokine production in vitro - Assessment of methodologies and cell types.

BACKGROUND: Hyperuricemia has been shown to be an inducer of pro-inflammatory mediators by human primary monocytes. To study the deleterious effects of hyperuricemia, a reliable and stable in vitro model using soluble urate is needed. One recent report showed different urate-dissolving methods resulted in either pro-inflammatory or anti-inflammatory properties. The aim of this study was to compare the effect of two methods of dissolving urate on both primary human peripheral blood mononuclear cells (PBMCs) and THP-1 cells. The two methods tested were 'pre-warming' and 'dissolving with NaOH'. METHODS: Primary human PBMCs and THP-1 cells were exposed to urate solutions, prepared using the two methodologies: pre-warming and dissolving with NaOH. Afterwards, cells were stimulated with various stimuli, followed by the measurement of the inflammatory mediators IL-1ß, IL-6, IL-1Ra, TNF, IL-8, and MCP-1. RESULTS: In PBMCs, we observed an overall pro-inflammatory effect of urate, both in the pre-warming and the NaOH dissolving method. A similar pro-inflammatory effect was seen in THP-1 cells for both dissolving methods after restimulation. However, THP-1 cells exhibited pro-inflammatory profile with exposure to urate alone without restimulation. We did not find MSU crystals in our cellular assays. CONCLUSIONS: Overall, the urate dissolving methods do not have critical impact on its inflammatory properties. Soluble urate prepared using either of the two methods showed mostly pro-inflammatory effects on human primary PBMCs and monocytic cell line THP-1. However, human primary PBMCs and the THP-1 differ in their response to soluble urate without restimulation.

Lipoxin A4 promotes antibiotic and monocyte bacterial killing in established Pseudomonas aeruginosa biofilm formed under hydrodynamic conditions.

Pseudomonas aeruginosa is a gram-negative, opportunistic bacteria commonly found in wounds and in lungs of immunocompromised patients. These bacteria commonly form biofilms which encapsulate the bacteria, making it difficult for antibiotics or immune cells to reach the bacterial cells. We previously reported that Lipoxin A4 (LxA4 ), a Specialized Pro-resolving Mediator, has direct effects on P. aeruginosa where it reduced biofilm formation and promoted ciprofloxacin antibiotic efficacy in a static biofilm-forming system. In the current studies, we examined the actions of LxA4 on established biofilms formed in a biofilm reactor under dynamic conditions with constant flow and shear stress. These conditions allow for biofilm growth with nutrient replenishment and for examination of bacteria within the biofilm structure. We show that LxA4 helped ciprofloxacin reduction of live/dead ratio of bacteria within the biofilm. THP-1 monocytes interacted with the biofilm to increase the number of viable bacteria within the biofilm as well as TNF-α production in the biofilm milieu, suggesting that monocyte interaction with bacterial biofilm exacerbates the inflammatory state. Pre-treatment of the THP-1 monocytes with LxA4 abolished the increase in biofilm bacteria and reduced TNF-α production. The effect of decreased biofilm bacteria was associated with increased LxA4 -induced monocyte adherence to biofilm but not increased bacteria killing suggesting that the mechanism for the reduced biofilm bacteria was due to LxA4 -mediated increase in adherence to biofilm. These results suggest that LxA4 can help antibiotic efficacy and promote monocyte activity against established P. aeruginosa biofilm formed under hydrodynamic conditions.

A macrophage-cell model of HIV latency reveals the unusual importance of the bromodomain axis.

BACKGROUND: Although macrophages are now recognized as an essential part of the HIV latent reservoir, whether and how viral latency is established and reactivated in these cell types is poorly understood. To understand the fundamental mechanisms of viral latency in macrophages, there is an urgent need to develop latency models amenable to genetic manipulations and screening for appropriate latency-reversing agents (LRAs). Given that differentiated THP-1 cells resemble monocyte-derived macrophages in HIV replication mechanisms, we set out to establish a macrophage cell model for HIV latency using THP-1 cells. METHODS: We created single-cell clones of THP-1 cells infected with a single copy of the dual-labeled HIVGKO in which a codon switched eGFP (csGFP) is under the control of the HIV-1 5' LTR promoter, and a monomeric Kusabira orange 2 (mKO2) under the control of cellular elongation factor one alpha promoter (EF1α). Latently infected cells are csGFP-, mKO2+, while cells with actively replicating HIV (or reactivated virus) are csGFP+,mKO2+. After sorting for latently infected cells, each of the THP-1 clones with unique integration sites for HIV was differentiated into macrophage-like cells with phorbol 12-myristate 13-acetate (PMA) and treated with established LRAs to stimulate HIV reactivation. Monocyte-derived macrophages (MDMs) harboring single copies of HIVGKO were used to confirm our findings. RESULTS: We obtained clones of THP-1 cells with latently infected HIV with unique integration sites. When the differentiated THP-1 or primary MDMs cells were treated with various LRAs, the bromodomain inhibitors JQ1 and I-BET151 were the most potent compounds. Knockdown of BRD4, the target of JQ1, resulted in increased reactivation, thus confirming the pharmacological effect. The DYRK1A inhibitor Harmine and lipopolysaccharide (LPS) also showed significant reactivation across all three MDM donors. Remarkably, LRAs like PMA/ionomycin, bryostatin-1, and histone deacetylase inhibitors known to potently reactivate latent HIV in CD4 + T cells showed little activity in macrophages. CONCLUSIONS: Our results indicate that this model could be used to screen for appropriate LRAs for macrophages and show that HIV latency and reactivation mechanisms in macrophages may be distinct from those of CD4 + T cells.

Imeglimin attenuates NLRP3 inflammasome activation by restoring mitochondrial functions in macrophages.

Imeglimin is a novel oral antidiabetic drug for treating type 2 diabetes. However, the effect of imeglimin on NLRP3 inflammasome activation has not been investigated yet. Here, we aimed to investigate whether imeglimin reduces LPS-induced NLRP3 inflammasome activation in THP-1 macrophages and examine the associated underlying mechanisms. We analyzed the mRNA and protein expression levels of NLRP3 inflammasome components and IL-1ß secretion. Additionally, reactive oxygen species (ROS) generation, mitochondrial membrane potential, and mitochondrial permeability transition pore (mPTP) opening were measured by flow cytometry. Imeglimin inhibited NLRP3 inflammasome-mediated IL-1ß production in LPS-stimulated THP-1-derived macrophages. In addition, imeglimin reduced LPS-induced mitochondrial ROS production and mitogen-activated protein kinase phosphorylation. Furthermore, imeglimin restored the mitochondrial function by modulating mitochondrial membrane depolarization and mPTP opening. We demonstrated for the first time that imeglimin reduces LPS-induced NLRP3 inflammasome activation by inhibiting mPTP opening in THP-1 macrophages. These results suggest that imeglimin could be a promising new anti-inflammatory agent for treating diabetic complications.

The in vitro gastrointestinal digestion-associated protein corona of polystyrene nano- and microplastics increases their uptake by human THP-1-derived macrophages.

BACKGROUND: Micro- and nanoplastics (MNPs) represent one of the most widespread environmental pollutants of the twenty-first century to which all humans are orally exposed. Upon ingestion, MNPs pass harsh biochemical conditions within the gastrointestinal tract, causing a unique protein corona on the MNP surface. Little is known about the digestion-associated protein corona and its impact on the cellular uptake of MNPs. Here, we systematically studied the influence of gastrointestinal digestion on the cellular uptake of neutral and charged polystyrene MNPs using THP-1-derived macrophages. RESULTS: The protein corona composition was quantified using LC‒MS-MS-based proteomics, and the cellular uptake of MNPs was determined using flow cytometry and confocal microscopy. Gastrointestinal digestion resulted in a distinct protein corona on MNPs that was retained in serum-containing cell culture medium. Digestion increased the uptake of uncharged MNPs below 500 nm by 4.0-6.1-fold but did not affect the uptake of larger sized or charged MNPs. Forty proteins showed a good correlation between protein abundance and MNP uptake, including coagulation factors, apolipoproteins and vitronectin. CONCLUSION: This study provides quantitative data on the presence of gastrointestinal proteins on MNPs and relates this to cellular uptake, underpinning the need to include the protein corona in hazard assessment of MNPs.

The evaluation of skin sensitization potential of the UVCB substance diisopentyl phthalate by in silico and in vitro methods.

Diisopentyl phthalate (DiPeP) is primarily used as a plasticizer or additive within the production of polyvinyl chloride (PVC), and has many additional industrial applications. Its metabolites were recently found in urinary samples of pregnant women; thus, this substance is of concern as relates to human exposure. Depending upon the nature of the alcohol used in its synthesis, DiPeP may exist either as a mixture consisting of several branched positional isomers, or as a single defined structure. This article investigates the skin sensitization potential and immunomodulatory effects of DiPeP CAS No. 84777-06-0, which is currently marketed and classified as a UVCB substance, by in silico and in vitro methods. Our findings showed an immunomodulatory effect for DiPeP in LPS-induced THP-1 activation assay (increased CD54 expression). In silico predictions using QSAR TOOLBOX 4.5, ToxTree, and VEGA did not identify DiPeP, in the form of a discrete compound, as a skin sensitizer. The keratinocyte activation (Key Event 2 (KE2) of the adverse outcome pathway (AOP) for skin sensitization) was evaluated by two different test methods (HaCaT assay and RHE assay), and results were discordant. While the HaCaT assay showed that DiPeP can activate keratinocytes (increased levels of IL-6, IL-8, IL-1α, and ILA gene expression), in the RHE assay, DiPeP slightly increased IL-6 release. Although inconclusive for KE2, the role of DiPeP in KE3 (dendritic cell activation) was demonstrated by the increased levels of CD54 and IL-8 and TNF-α in THP-1 cells (THP-1 activation assay). Altogether, findings were inconclusive regarding the skin sensitization potential of the UVCB DiPeP-disagreeing with the results of DiPeP in the form of discrete compound (skin sensitizer by the LLNA assay). Additional studies are needed to elucidate the differences between DiPeP isomer forms, and to better understand the applicability domains of non-animal methods in identifying skin sensitization hazards of UVCB substances.

Fusobacterium nucleatum dysregulates inflammatory cytokines and NLRP3 inflammasomes in oral cells.

OBJECTIVE: This study aimed to clarify the difference in Fusobacterium nucleatum (F. nucleatum) induced inflammatory cytokines and nod-like receptor protein 3 (NLRP3) inflammasomes dysregulation among three periodontal cells. METHODS: Oral epithelial cells (HIOECs), THP-1 macrophages, and human gingival fibroblasts (HGFs) were exposed to F. nucleatum with/without adenosine triphosphate (ATP) and nigericin (Nig). Cell morphology was assessed by scanning electron microscopy. qRT-PCR, protein microarrays, and bioinformatic methods were used to evaluate the cytokines and their complex interplay. NLRP3 inflammasomes activation was detected by western blotting and ELISA. RESULTS: F. nucleatum adhered to and invaded cells. In HIOECs, F. nucleatum enhanced interleukin (IL)-1α/1ß/6/10/13, TNF-α, and interferon (IFN)-γ expression. In THP-1 macrophages, F. nucleatum up-regulated IL-1α/1ß/6/10 and TNF-α levels. In HGFs, F. nucleatum increased IL-6 levels. F. nucleatum and ATP synergistically boosted IFN-γ level in THP-1 macrophages and IL-13 level in HGFs. IL-1α/1ß/6, and TNF-α served as epicenters of the inflammatory response. Additionally, F. nucleatum activated NLRP3 inflammasomes in HIOECs, and ATP/Nig boosted the activation. F. nucleatum also triggered NLRP3 inflammasomes in THP-1 macrophages, but in HGFs, only NLRP3 and caspase-1 levels were elevated. CONCLUSION: F. nucleatum infiltrated periodontal supporting cells and dysregulated inflammatory cytokines and NLRP3 inflammasomes.

Elucidating miR-146a-3p as a key player in autophagy and lipid metabolism in Leishmania major infection.

Autophagy is a key step involved in many unicellular eukaryotic diseases, including leishmaniasis, for cellular remodelling and differentiation during parasite's lifecycle. Lipids play a significant role in the infection process that begins with Leishmania major invading host cells. MicroRNAs (miRNAs), a family of small, 22-24 nucleotide noncoding regulatory RNAs, target mRNAs to modify gene expression and, subsequently, proteome output may have a regulatory role in altering the host cell processes. We observed miR-146a-3p expression increases in a time-dependent manner post Leishmania major infection. Transfecting miR-146a-3p mimic increases the expression of ATG7, an autophagy gene that encodes an E1-like enzyme in two ubiquitin-like conjugation systems required for autophagosome progression. HPGD (15-hydroxyprostaglandin dehydrogenase) operates as an enzyme, converting prostaglandin to its non-active form. Microarray data and western studies reveal that miR-146a-3p targets and inhibits HPGD, thereby increasing prostaglandin activity in lipid droplets. Herein, our research focuses on miR-146a-3p, which boosts ATG7 expression while reducing HPGD post Leishmania major infections helping us comprehend the intricate network of microRNA, autophagy, and lipid metabolism in leishmaniasis.